Injectable pharmaceutical preparation, and a method of making same



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3,tl3,8l5 INJECTABLE PHARMAtIEUTICAL PREPARATION, AND A METHOD OF MAKINGSAME Hans Licb, Universitaetsplatz 2, Graz, Austria; Ernst Kupelwieser,Moenchsberg 17, Salzburg, Austria; and Anton Holasck, Universitaetsplatz2, Graz, Austria No Drawing. Original application Oct. 11, 1951, Ser.No. 250,961, now Patent No. 2,961,374, dated Nov. 22, 196i). Dividedandthis application Oct. 26, 1960, Ser. No. 65%? Claims priority,application Austria Oct. 14, 1950 15 Claims. (Cl. 16758) The presentinvention relates to injectable pharmaceutical preparations capable offorming, on injection,

depots of therapeutically effective compounds, and amethod of makingsame.

The present application is a division of copending application SerialNo. 250,961, filed October 11, 1951, and entitled InjectablePharmaceutical Preparation, and a Method of Maleing Same, now Patent No.2,961,374.

In therapy, it is ofiten of great importance that therapeuticallyeffective compounds, when administered parenter-ally, have a prolongedactivity. In order to achieve this purpose, a depot of thetherapeutically effective compound is produced in the body by injectionat the place of injection, said depot allowing continuous but retardedabsorption and, thus, continuous but prolonged action of thetherapeutically effective compound.

However, it was found to be very difficult to maintain a sufiicientlyhigh blood level of such compounds over a prolonged period of timewithout causing overdosing. In order to produce such depots, variousmethods were suggested heretofore. For instance, difiicultly solubledrugs were implanted in the form of pellets or the like into the body.Readily soluble drugs were coated, before implantation, with coatingswhich rendered their absorption by the body fluids more difiicult. Suchcoatings consisted of Waxes and the like. This manner of producing adepot of a therapeutically effective compound in the body has severaldisadvantages. First, implantation necessitates a bloody operation withall the dangers of infection and the like connected therewith.Frequently, the implanted pellets, soon after implantation, weresurrounded by tissue and are, so to say, encased in the form of a cyst,thus preventing further absorption of the therapeutically effectivecompound.

Another method of producing depots of therapeutically effectivecompounds consists in injecting such a compound in the form of asuspension in suitable liquids. However, when using water solublecompounds, and injecting, tor instance, their suspensions in oils andthe like, the retarding effect of such injections is usually very shortand it is often difficult to avoid overdosing and the reaction of anexcessively high blood level shortly after injection and of too low ablood level later on.

It has also been suggested to administer the therapeutically effectivecompound in the form of an emulsion or dispersed in a viscous colloidalvehicle, said emulsions and vehicles being adapted to retard absorption.Proca-ine penicillin, for instance, has been administered in suspensionin an oily vehicle to which aluminum stearate was added. Thereby athixotropic medium is formed which prevents too rapid absorption of thepenicillin salt. But such thixotropic additions cause only a slight andtemporary increase in viscosity and do not form a depot of sufiicientlyprolonged activity. Such suspensions have furthermore the disadvantagethat they tend to clog the injection needle. Therefore, injectionneedles of large diameter have to be used which cause pain on insertingthem into the body.

Mixtures of polyvinyl alcohols or their water-soluble assists PatentedMay 14, 1963 derivatives with at least one solvent capable of dissolvingsaid polyvinyl compounds and with the addition of Congo red, have alsobeen suggested as carriers for the therapeutically effective compounds.Such mixtures are liquid at about 45 C. Such preparations were injectedat 45 C. and solidified at body temperature. The injection of such awarm preparation is quite disagreeable. Furthermore, since suchsolidiiied polyvinyl compounds are water-soluble, even in the solidstate, they are very readily dissolved by the body fluids and absorbed.Therefore, the prolonged efi'ect achieved by using such a carrier for atherapeutically eflective compound is comparatively short.

The method of implanting pellets and the like by surgical operationshould be avoided if possible, especially if the therapeuticallyeffective compound has to be administered repeatedly. Furthermore, saidcompounds, on account of their decreased solubility when compressed totablets, pellets, and the like, very often are not sufficiently solubleto produce the desired therapeutic effect.

Emulsions and colloidal vehicles have the disadvantage that onlyemulsions and vehicles having a limited range of viscosity can be usedfor the production of depots because diificulties in injecting suchemulsions and vehicles are often encountered. Even when using syringeswith needles with a very Wide diameter, only such vehicles are suitablefor injection which are still liquid and fiowable.

With thixotropic vehicles, only slight and labile changes and increasesin viscosity are produced, due to the nature of the thixotropicphenomenon. The vehicles do not solidify as is necessary to producedepots of long duration.

One object of the present invention is to provide depot vehicles fortherapeutically effective compounds which depot vehicles allow injectionby means of ordinary syringes and needles of normal diameter and,thereby, production of depots which are dissolved mainly by fermentativeor cellular effects.

Another object of this invention consists in providing pharmaceuticalpreparations -for parenteral administration which, on injection, producea prolonged effect of the therapeutically active compound contained insaid preparation but which does not possess the disadvantages of theknown and heretofore used depot preparations as mentioned above.

ther objects of this invention will become apparent from thespecification and the examples illustrating the invention more indetail.

The invention consists in principle in combining at least two watersoluble components which may be liquid or sol-id compounds or may beemployed in solution and which are capable of reacting with each other,or influencing each other, in such a manner that the solubility of thereaction product in water is reduced and the reaction productsolidifies. Such proportions of said components are used that theinjectable solution obtained by such a combination, initially, is liquidbelow or at body temperature but solidifies to a difliculty solubledepot within a predetermined period of time. The two or more componentsare combined with each other, preferably immediately before injection,in such a manner that they solidify in the body after a predeterminedperiod of time, as planned. Said components must be substantiallynontoxic and non-irritating to and well tolerated by the body, in theconcentrations and amounts employed. Their reaction product forming thedepot vehicle must be slowly but completely absorbed from and eliminatedby the body.

In order to carry out the process of this invention, there is firstprepared a solution of a coagulable, setting, or in any other mannersolidifying material as well as a solution of a material capable ofinducing or causing such coagulation, setting, or solidification, forinstance, a solution of a coagulating or hardening agent. Theconcentration of said solutions are adjusted with respect to each otherin such a manner that, after combining the same, the materials containedtherein solidify after a predetermined period of time and form adifiicultly soluble depot within the area of administration to the body.Examples of such systems are, for instance: Fibrinogen-thrombin,gelatin-formaldehyde, gelatin-quinone, gelatin-cobalt compounds,gelatin-chromium compounds, and many others. Of course, it is possibleto use at the same time more than one hardening agent and/or more thanone solidifying material. The therapeutically effective compound may bedissolved or dispersed in any one or in all of these components. It mayalso be added to the solution obtained after combining the twocomponents, just before injection.

The injection solution may be rendered less viscous by various methods,such as a slight heating, addition of compounds capable of regulatingviscosity, and the like. Of course, only such additives should be usedwhich do not substantially impair subsequent solidification of thecomponents.

The process according to the present invention and the preparationsobtained thereby may be used, among others, for producing depots ofanalgesics, local anesthetics, antibiotics, agents stimulating thevegetative nervous system, hormones, especially insulin, parathyroidhormone, pituitary gland hormones, agents to produce controlledhypothermia agent useful in the treatment of parkinsonism antiepilepticagents, antihistaminic agents, prolylactic and therapeutic agentsadministered in the prevention and treatment of apoplexia, and others.

According to this invention, a vehicle for a therapeutically efiectivecompound is used which, before injection, is in the liquid state butwhich, after injection, solidifies at the place and area of injection toa ditficultly soluble, solid, for instance gel-like material, saidmaterial retaining dispersed therethrough the therapeutically effectivecompound so that its absorption is retarded and that it exerts aprolonged effect. Dissolution and absorption of the therapeuticallyeffective compound from said solidified vehicle takes place either bydiffusion or by dissolution at a rate at which the vehicle itself isdissolved and absorbed by the body.

One of the components may be used in solid form and the other in theform of a solution. The components may also be mixed in solid dry formand may be dissolved shortly before injection so as to yield thesolidifying or, respectively, gelling mixture. The components may besolidified in the body by coagulation but also other types ofsolidification may be used. Especially suitable is a system of compoundswhich solidifies by hardening and sets to a mass which is relativelyinsoluble in body fluids.

A coagulating system suitable for carrying out this invention is thesystem fibrinogen-thrombin. Fibrinogen, thrombin, and a preferablydifiicultly soluble therapeutically effective compound, in the drystate, are placed into a vial. The amount of thrombin is calculated insuch a manner, that a solution of the contents of said vial, preferablya physiological salt solution, requires, at a temperature of 37 C.,about 4 minutes to 5 minutes for coagulating the fibrinogen solution.For administering said fibrinogen-thrombin drug mixture, the contents ofsaid vial are mixed with the necessary amount of physiological saltsolution whereby a suspension of the medicament in thefibrinogen-thrombin solution is obtained. On account of thecomparatively low temperature of the salt solution the coagulation timeis somewhat retarded. But on injecting such a mixture and suspensioninto the body, the fibrinogen very soon forms a fibrin gel which keepsthe drug in an adsorbed condition and prevents its rapid ab sorption bythe body fluids. However, after a certain period of time the fibrin alsois gradually and completely absorbed by the body thereby graduallyreleasing the drug which is slowly absorbed at the same rate. Thefibrin, as long as it is present in the body, prevents too rapid anabsorption of the drug because it forms a comparatively firm combinationtherewith. Only after the fibrin molecule has been broken down will thedrug be absorbed because it is then exposed to the action of the bodyfluid. It is possible, for instance, to produce by this method prolongednerve block anesthesia by means of dibucaine hydrochloride(Z-butoxy-N-(Z-diethylamino ethyl) cinehonine amide hydrochloride). Thiscompound, as is known, is many times more effective than procainehydrochloride but also more toxic. By the method of this invention,larger amounts of said dibucaine hydrochloride may be administered thuscausing a more prolonged anesthetic efiect without the danger ofdisagreeable side-effects, such as necrosis of tissue, gangrene or, inlower concentrations, of slight temporary vascular dilatation.

Another very suitable system which is preferably employed to carry outthis invention is the system gelatinformaldehyde. It is advisable toemploy for the purpose of this invention a gelatin of a melting point ashigh as possible. The suitability of a gelatin may be determined in asimple and effective manner by preparing an 18% aqueous solution ofgelatin, adding 0.2 cc. of a 2.5% formaldehyde solution to 2.0 cc. ofsaid 18% gelatin solution, mixing the mixture thoroughly by means of aglass rod, and determining the gelling time at 37 C. The better thegelatin, the quicker does the solution gel. Such 18-20% gelatinsolutions are, even at 37 C., rather viscous. Their viscosity andmelting point is lowered considerably by the addition of potassiumthiocyanate, for instance, in amounts of 35%. A further reduction ofviscosity is achieved by the addition of calcium ions which aresupplied, for instance, in the form of an 0.7% calcium chloridesolution.

Since formaldehyde has a slightly irritating effect, care must be takento use as little thereof as possible. This is achieved by the use ofgelatin solutions of high concentration. Addition of a small amount ofan alkali metal hydroxide has proved of value. For instance, theaddition of 1 cc. of a N/lO sodium hydroxide solution to 2 g. of gelatincauses buffering of the gelatin solution and the irritating eifect offormaldehyde is diminished while the pH-value of the injection-solutiondoes not exceed a pH of about 8.0. When preparing, for instance, aninsulin composition of prolonged activity, such a small addition ofsodium hydroxide does not impair its activity. Apparently the insulin isprotected by the gelatin. The higher the alkali metal hydroxideconcentration, the lower the amount of formaldehyde required forhardening the gelatin.

Although formaldehyde addition shortly before injection apparentlyprotects the gelatin solution against contamination by infectivemicroorganisms, it is nevertheless advisable to subject the gelatinsolution to sterile filtration, for instance, by means of a Seitz filterand the like. A highly concentrated gelatin solution (IS-20% however,can not be filtered through such a filter. There fore, a dilute solutionof, for instance, 1012% gelatin is filtered and is subsequentlyconcentrated by evaporation in a vacuum.

The formaldehyde solution used contains preferably about 1% offormaldehyde. 0.2-0.3 cc. are added to 2 cc. of an 1820% gelatinsolution. The formaldehyde solution is preferably stabilized. This canbe done, for instance, by the addition of sodium chloride. Amounts of 5%to 10% of sodium chloride have given satisfactory stabilizing effects.

When adding formaldehyde to a slightly alkaline gelatin solution, a gelis formed immediately at the places of contact of the gelatin and theformaldehyde solution.

sodium chloride.

In order to avoid such premature gel formation, hydrochloric acid isadded to said formaldehyde solution. Gel formation takes place onlyafter said acid is neutralized, i.e. as soon as both solutions have beenmixed thoroughly. The formaldehyde solution contains preferably about0.365 g. of hydrochloric acid in 1000 cc., corresponding to an N/ 100hydrochloric acid solution.

As has been found, best results are obtained by using a formaldehydesolution containing about 1% of formaldehyde, 0.0365% ofhydrochloricacid, and 5% to 10% of Such a formaldehyde solution is keptin tube-like graduated ampoules provided with rubber closure caps whichallow easy withdrawal of amounts of 0.2 cc.

Gelatin and formaldehyde concentration have a considerable influenceupon gelling time as is evident from the following Table 1:

Table 1 Gelling time, in minutes, of a gelatin solution containing zFormaldehyde per 0.2 cc. percent of gelatin 4.5 2.5 7 M9.() 6.8 1 ore25. 2mg than 60 When adding sodium hydroxide to the gelatin solution,the amount of formaldehyde necessary to produce a gel within areasonable period of time is considerably reduced as is evident from thefollowing Table 2:

Table 2 2 cc. of a 23% gelatin solution to which the below given amountsof sodium hydroxide were added, were hardened by the addition of 0.2 cc.of a 4% formaldehyde solution within the following period of time:

Sodium hydroxide addition expressod'in normality 0045 0. 0023 0. 0011 0.0006 Gelling time in minutes 1. 5 3.5

2 cc. of a 10.7% gelatin solution containing sodium hydroxide in anamount sumcient to form an 0.0045 N solution, yield with 0.2 cc. of a 1%formaldehyde solution a honeylike mass within 21 minutes.

2 cc. of a 20% gelatin solution of 0.01 normality in sodium hydroxideform with 0.5 cc. of an 0.3%

formaldehyde solution a gel within 8 minutes.

cc. of a 20% gelatin solution of 0.01 normality in sodium hydroxide formwith 0.5 cc. of an 0.2%

formaldehyde solution a sufiiciently firm gel within 25 minutes. After16 minutes the mixture has the consistency of honey.

The gelatin and the formaldehyde solutions may be mixed either in theampule containing the gelatin solution or in a syringe. When mixing inthe amopule, it is advisable to add the formaldehyde solution to thegelatin solution and then rapidly mixing said solutions. When mixing inthe syringe, first the gelatin solution is drawn up and thereafter theformaldehyde solution. Both solutions are then thoroughly mixed by meansof an air bubble. However, on account of the high viscosity of thegelatin solution, very frequently this method of mixing does not givesatisfactory results. Therefore, a metal ball is placed into the syringethe movements of which cause the two solutions to be readily,completely, and rapidly mixed with each other. The piston of the syringeis preferably provided with a recess so as to receive the metal ball andto allow quantitative injection of the contents of said syringe. Inorder to prevent adhesion of the ball to the piston, a small spring isprovided at the bottom of the recess of said piston.

The retarding eifect of a preparation according to this invention isreadily demonstrated by the following experiment:

The dyestuif fluorescein, after administration to a guinea pig, isexcreted almost quantitatively in the urine and can readily be detectedby the fluorescence it imparts to urine, said fluorescence beingvisual-1y observable. 2 cc. of a solution of fluorescein in water,containing 0.37 g. of said dyestuif in cc., were subcutaneously injectedinto control animals. The animals were kept on a green fodder (herbage)diet so as to produce large amounts of urine. Their urine was collectedevery hour. It was found that urine collected after 5 hours to 7 hoursdid not show any fluorescence on observation in daylight. This indicatesthat all the fluorescein was excreted within 5 hours afteradministration.

2 cc. of a 23% gelatin solution containing 0.37% of fluorescein andmixed, before injection, with 0.2 cc. of a 2.8% formaldehyde solution,was injected subcutaneously into another animal. fter 1 hour,fluorescence of the urine could be detected indicating that excretion ofthe dyestuif had already started. Fluorescence did not markedly diminishWithin the first 31 hours. Even after 44 hours, weak fluorescence couldbe detected, thus indicating that the depot produced by injecting apreparation according to this invention has a very remarkable prolongingeffect.

A 16% gelatin solution and a 20% gelatin solution of the dyestuif weremixed with a formaldehyde solution as described above. The mixturesobtained thereby were administered, by intramuscular injection, to twoother guinea pigs. The urine of these animals showed also strongfluorescence up to 29 hours whereafter it slowly disappeared.

in another experiment, a solution of fluorescein sodium in a 20% gelatinsolution was intramuscularly injected into a rabbit. The urine did notexhibit any fluorescence after 7-h'ours, thus indicating that all thedyestuff was excreted. The same amount of a 20% gelatin solutioncontaining the same amount of fluorescein but mixed, before injection,with a small amount of a 1% formaldehyde solution was injected intoanother rabbit. Even after 24 hours marked excretion of fluoresceincould be detected in the urine.

These experiments prove that the depot eifect of preparations accordingto this invention is quite remarkable. The dyestuif, although releasefrom its depot starts a short time after injection, remains in the bodyabout seven times longer than on administration in aqueous solution.

It is possible to combine the gelatin-formaldehyde depots with oilemulsions, i.e. to use oil emulsions in gelatin solutions which are thenhardened by formaldehyde in the same manner as described with aqueousgelatin solutions. Thus, therapeutically effective compounds can beadministered which are soluble in oils but are not soluble in water. Inthis case it is, however, advisable to somewhat reduce the viscosity ofthe gelatin solution. The amount of formaldehyde added need not bechanged because, as far as has been found, it depends merely upon theamount of gelatin present in such preparations.

Intramuscular application apparently does not produce depots suchprolonged activity as depots produced by subcaneous injection.

Another important application of depot preparations according to thisinvention consists in their use for filling body cavities and cavitiesproduced by diseases, such as abscesses, bone cavities, tubercularcaverns, and the like, with therapeutically effective compounds. It isknown to drain and rinse such cavities by means of suitable medicamentedsolutions or to spray them with a medicamented powder. But suchsolutions and powders remain therein only for a short period of timeand, therefore, have only a brief therapeutic effect. It is of greatimportance to provide preparations which, after introduction into suchcavities, remain therein for a longer time. This is possible by using adepot preparation according to this invention and by filling the cavitytherewith. Since the preparation is administered in the liquid state andsolidifies only after introduction into the cavity, it is capable offilling out the entire cavity, whereafter it is converted into the solidstate. Thereby, not only the advantage is achieved that the cavity ismechanically closed and sealed for a prolonged period of time, but thatit is possible to introduce drugs in amounts which cannot beadministered by other methods because, due to their rapid absorption,they would have a detrimental effect upon the body. A preparationaccording to this invention, however, does not release the drug rapidlyand, therefore, much larger amounts of drugs, antiseptics, and the like,may be applied to the cavities. Since the cavity, in general, is notexposed to the action of the body fluids to such an extent as, forinstance, the muscle, the further advantage is achieved that actuallydissolution of the drug depot takes place at a comparatively slow rate,thereby further extending the effect of the drug.

A preparation according to this invention may also be injected into theuterus or into the bladder and the like. The new preparations,especially those containing the fibrinogen-thrombin system mayfurthermore be used for obliteration of varicose veins. It is evidentthat such new preparations may find extensive application in therapy incases where prolonged activity of a medicament is essential ordesirable.

Preparations of the type gelatin-quinone have proved to be especiallyeffective, but other systems may be used likewise.

Fibrinogen may also be used for the purpose of this invention.Fibrinogen obtained from human blood is preferred. It may be preparedaccording to directions given, for instance, by Cohn, Strong, Hughes etal. in Journ. Am. Chem. Soc., vol. 68, page 459 (1946), or by Wunderlyin Helv. Chim. Acta, vol. 31, page 49 (1948).

Thrombin may be produced according to the instructions given by Seegerset al., Journ'. Biol. Chem, vol. 123, page 751 (1938). The time ofcoagulation of fibrinogen by thrombin is influenced by the fibrinogenconcentration, the thrombin concentration, the ion content of saidsolutions, the type of ions present, the presence of proteins, thepH-value, and others more. It may be retarded by various additions suchas glycerol, salicylates, benzene sulfonates, and others. Increase infibrinogen concentration while the thrombin concentration and all otherconditions remain constant, increases coagulation time. Increase inthrombin concentration decreases coagulation time. Increase in ionconcentration also increases coagulation time, whereby the type of ionspresent is of great importance. Some ions, such as citrate, phosphate,and the like ions, are capable of considerably retarding coagulation,even when present in small concentrations. Chloride ions (for instance,addition of sodium chloride to the fibrinogen solutions) also retardcoagulation with increasing amounts. On the other hand, other ions,especially calcium ions, are capable of accelerating coagulation withincreasing amounts. Contamination by proteins which, of course, shouldbe avoided, especially when administering the new preparations to humanpatients, retards coagulation. Change in hydrogen ion concentration hasalso a remarkable influence upon coagulation. Neutral solutions showshortest coagulation time. With increasing pH-values and even more withdecreasing pH-values, coagulation time increases. Glycerol has astrongly retarding effect upon coagulation. Temperature increases up to30 C. cause decrease in coagulation time while above 30 C. coagulationis gradually retarded.

Fragility and elasticity of the resulting fibrin gel is closelyconnected with its power of retraction. Fragile and, therefore, lessretractable fibrin gels are obtained by using higher fibrinogenconcentrations. Addition of retarding agents, such as glycerol, is alsocapable of producing less retractable gels. It is, therefore, advisable,when using higher thrombin concentrations, to employ higher fibrinogenconcentrations and to add retarding agents in order to prolongcoagulation time. The onkotic pressure of the fibrin gel, thus, can beregulated by the addition of salts, colloids, such as gelatin, andothers, so that it does not dilfer too much from that of the tissue.

The system fibrinogen-thrombin can be adjusted in such a manner that itis possible to produce compositions of different coagulation time. Forinstance, when increasing the fibrinogen concentration but keeping allother conditions constant, the coagulation time increases as will beseen from the following Table 3:

Table 3 Coagulation time in minutes Fibrinogen concentration, percent;

0.5 units of 2 units of thrombin thrombin per cc. pmcc.

That increase in thrombin concentration decreases coagulation time, isshown by the following Table 4.

The influence of ion addition to the fibrinogen-thrombin system is shownin the following Table 5 whereby sodium chloride is added to thefibrinogen-thrombin solution:

Table 5 Sodium chloride concentration Coagulation time expressed innormality: in minutes The effect of hydrogen ion concentration uponcoagulation time is demonstrated in the following Table 6. It isshortest at a pH of 7.0, but increases with decreasing pH-values.

Table 6 pH: Coagulation time in minutes 6.9 2.1 6.6 3.2 6.3 5.0

The retarding effect of glycerol is shown in the following Table 7.

Table 7 Glycerol addition Coagulation time in percent: in minutes Theinfluence of temperature increase is shown in the following Table 8.

Table 8 Temperature Coagulation time in C.: in minutes In the place ofthrombin, there may be used other substances to cause coagulation offibrinogen, for instance, ninhydrin (triketo hydrindene hydrate),salicylic aldehyde, alloxan, and others. Although ninhydrin has sometoxic effect, it is used in such small amounts (0.l1.() mg. per 1 g. offibrinogen) that it cannot exert any unfavorable effect upon the body.

Since fibrinogen and thrombin are stable only in the solid dry state,the best way to store a fibrinogen-thrombin preparation according tothis invention is to keep fibrinogen in an ampoule, thrombin in anotherarnpoule, and the aqueous solution which may contain the accelerating orretarding agents, buffering compounds, and the like, in a third ampoule.Shortly before injection fibr'mogen is first dissolved whereafter thefibrinogen solution is added to thrombin, and, after thorough mixing,the mixture is injected into the body. The therapeutically elfectivecompound may be added to the fibrinogen or to the thrombin or it may bedissolved or suspended in the solvent solution or it may even be addedafter mixing fibrinogen and thrombin. It is, of course, also possible toplace fibrinogen, thrombin, and the drug into the same ampoule,especially if a fibrinogen is selected which has about the samesolubility properties as thrombin, and to add thereto the solventsolution, whereafter the mixture is thoroughly mixed until all thefibrinogen and thrombin' are dissolved; The solution or suspension ofthe drug in said fibrinogen-thrombin solution can then be injected.

Formaldehyde, for gelatin hardening, is used in such small amounts thatno toxic effects need be feared. Furthermore, substantially all of saidformaldehyde is combined With the gelatin.

Chromium -compounds can also be used as gelling agents in very smallamounts so that no toxic effect will be caused. It is advisable, whenusing chromium com pounds as hardening agents, to add local anestheticsto the preparation because usually injections of chromium compounds aresomewhat painful.

Chromium ions are added by using a solution of a suitable chromium salt,such as chrome alum in the form of a 1% solution corresponding to an0.1% solution of chromium ions. Such a dilute solution, however, must bestabilized, for instance, by the addition of neutral salts, in order toretard hydrolysis. Acid addition is also capable of increasing thestability of such solutions; but a gel produced by means of such anacid-stabilized solution liquefies after a few hours. Therefore, such asolution can be used for very specific purposes only. The hardening timeis shortened with increasing chromium ion con.- centration.

As cobalt compounds there are used with great success cobaltitriaminecomplex compounds. They are without toxic effect when applied in thesmall amounts required to cause hardening of gelatin.

When using cobalt in the form of the so-called dichro 24 hours durationare obtained. When adding sympaammino cobaltichloride of the formula [CO3.1120 .Uz]

it is necessary to retard hardening because a gel forms immediately onaddition of said compound. For this purpose, potassium thiocyanate isadded to the gelatin solution. The more potassium thiocyanate ispresent, the more slowly proceeds the hardening process and the moredichro chloride is necessary. Adding acids, such as hydrochloric acid,to the dichro chloride solution also retards gelling, but the gel formedliquefies within a comparalid tively short time as With chromiumcompounds. Other cobalt triammine complex compounds than the dichrochloride may also be used. All these cobalt complex salts, however, formrather unstable solutions. Therefore, such solutions have to be freshlyprepared and must be stored in the dark, in order to retard hydrolysis.Hydrolysis of such solutions may be suppressed for a few days by theaddition of ammonium chloride, gum arable, organic dyestuffs, such asmethylene blue and the like.

Aluminum compounds may also be used as hardening agents. They formimmediately after addition, a gel with gelatin. Therefore, gel formationretarding agents must be added. Aluminum compounds are especiallysuitable on account of their very low toxicity.

Hardening by metal compounds, generally, does not produce depots of verylong duration. Usually depots of 24 hours duration are obtained. Whenadding sympathomimetic agents, such as adrenalin, synephrin tartrate,and other compounds of this type, to preparations containing metalcompounds as hardening agents, the flow of body fluid is reduced and,therefore, less drug is absorbed due to contraction of the bloodvessels.

Quinone is capable of forming a very stable gel with gelatin whichremains in the body for a very long time. The small amounts of quinoneused (about 10 mg. of quinone for 2 cc. of gelatin solution) areharmless.

Such quinone-gelatin depots are very hard and are slowly dissolved bythe body fluids when injected subcutaneously. Their prolonged eifect maylast for several weeks. Another advantage of quinone is that it causeshardening of relatively dilute gelatin solutions, such as solutionscontaining only 12% of gelatin. Such solutions are quite liquid and,therefore, are especially suitable for injection. One disadvantage ofquinone-hardened gelatin gels is their discoloration. The solutionstarts to acquire a yellowish to brownish color shortly after additionof the quinone. It is also possible to add quinone in solid form togelatin solutions because it is readily dissolved by such solutions.Like quinone there maybe used quinhydrone which requires, of course,larger amounts.

It is possible to combine various hardening and coagulating agents, suchas, for instance, chromium compounds and formaldehyde. The chromiumcompound forms the gel rapidly While the formaldehyde has a much slowerhardening effect, especially when the gelatin solution is not alkaline,but yields a hard gel. Such a mixture of hardening agents is especiallysuitable for hardening a slightly acid gelatin solution.

Such slightly acid gelatin solutions are often required because variousdrugs are not stable in alkaline gelatin solutions. An acid gelatinsolution, however, requires for hardening a very long time, often days.In such a case the use of a mixture of hardening agents of rapid action,such as a chromium salt solution, and of slow action, such asformaldehyde, has been of great advantage. it is, of course, alsopossible to add a slightly alkaline formaldehyde solution, which,however, is not very stable, or an alkaline formaldehyde compound to thegelatin solution. One may also add formaldehyde to the gelatin solutionand thereafter start the hardening process by the addition of a buffersolution capable of producing a slightly alkaline reaction. Of course,the gelatin solution should not become too acid in order to avoid anyhydrolytic decomposition thereof.

Or" course, other hardening and tanning agents may also be used for thepurpose of this invention although formaldehyde represents the preferredhardening agent.

The therapeutically efiective compound and the two components, namelythe coagulating or solidifying agent and the material capable ofsolidifying or coagulating under the influence of such agents, are mixedand combined, shortly and immediately before injection, by the physicianor the person administering the new preparation. In the case of gelatin,it is advisable to heat the gelatin solution to about 3035 C.solidification starts already in vitro. it must be retarded to such anextent that injection of the solution by an ordinary injection needle ispossible. This increase in viscosity of a gelatin solution to whichformaldehyde Was added, may be observed in vitro by inserting a glassrod into the mixture and withdrawing said glass rod after a certainperiod of time. In all cases, a filament-like structure adheres to theend of the rod, said filament becoming thicker and thicker withincreasing viscosity. Finally the viscosity is so high and hardening hasproceeded to such an extent, that the entire viscous content togetherwith the test tube can be lifted when trying to withdraw the glass rod.The gel, thereafter, does not change its form and surface.

A gelatin-formaldehyde mixture according to the present invention isinjected when the viscosity has not yet increased considerably, usuallyimmediately after mixing of the components. Concentration of thecomponents and other additions and conditions are adjusted in such amanner that the gel is formed within 6 minutes to 25 minutes. It hasbeen found that such a mixture, on injection, although the gel is notformed, has attained such a viscosity that almost none of the drug isdissolved and removed by the body fluids after injection. Addition ofadrenalin or a compound of similar activity to prevent prematureabsorption of the drug due to its contracting effect upon the bloodvessels, is of advantage. Gelatin of low molecular weight may also beused. Such a gelatin is, for instance, obtained by degradation ofcommercially available gelatin. Such a gelatin of lower molecularweight, for instance, of a molecular weight of 20,000 is converted intothe desired depot gel by the addition of formaldehyde or other hardeningagents to which accelerators such as polyvalent phenols, for instance,phloroglucinol are added.

It is, of course, possible to prepare combinations according to thisinvention, which allow the physician to wait for 10 minutes to 30minutes after mixing, before injecting the mixture. But it had provedthe preferred procedure to inject the mixture immediately after mixingand, therefore, to adjust concentration and other condi tionsaccordingly.

Potassium tlr'ocyanate is the preferred agent for regulating theviscosity of the gelatin solutions, but other agents, such as magnesiumchloride, calcium chloride, and others, may be used likewise.

Small amounts of calcium ions (-08%) or larger amounts of magnesium ionshave the same reducing effect upon the viscosity. Magnesium ions are ofadvantage because they can be injected in very large amounts withoutcausing any side-effects. On the contrary, magnesium exerts a good localanesthetic effect and, therefore, is capable of eliminating anyirritation caused by the hardening agent. Organic compounds, such asfurfuryl alcohol in amounts of 25%, are also capable of reducing theviscosity of gelatin solutions.

Iodine ions may also be used; but, of course, they are not entirelyharmless. Potassium cyanate may be added in amounts up to about 5%,calcium chloride in amounts up to 4%, magesium sulfate or magnesiumchloride in amounts up to 50%.

In place of formaldehyde, it is possible to employ formaldehyde whichhas already reacted with gelatin. Such a material is obtained, forinstance, by treating a dry gelatin powder in the cold with aformaldehyde solution (2% formaldehyde), drying the treated gelatin in avacuum, and comminuting the dried product to a powder. When adding sucha powder to a gelatin solution, the gelatin therein is also hardened.

Formaldehyde may also be used in the form of a solid compound whichyields free formaldehyde under the influence of a gelatin solution or ofchanges in the reaction conditions, for instance, by a rise intemperature. As such formaldehyde compound there may advantageously beemployed, for instance, anhydromethylene citrate which splits offformaldehyde at a temperature of 37 C. it is even possible to place asolid formaldehyde compound which is stable in the dry state and a drygelatin powder into the same ampoule and to add an aqueous solution orsuspension of the drug to said dry mixture to dissolve the gelatin andthe formaldehyde compound and to cause hardening.

When using coagulating or hardening agents which might react with thetherapeutically effective compound, it is advisable to employ saidcompounds in a solid state or in a diflicultly and slowly soluble form.For instance, when using insulin as therapeutically effective compound,it is preferably employed in the form of its difiicultly solublecompounds, such as protamine-insulin, hexamethylenetetramine-insulin,and the like. Formaldehyde will then react much quicker with the freeamino groups of the gelatin than with the insulin complex.

The system fibrinogen-thrombin may be used for most drugs. Of course,when administering, for instance, heparin or the like compounds whichprevent coagulation of the fibrinogen-thrombin system, it is advisableto use other coagulating agents, such as ninhydrin or salicylicaldehyde, in the place of thrombin.

When using the system fibrinogen-thrombin, the depot formed remains inthe tissue for a very long period of time provided the drug is added inthe form of a colloidal solution, a suspension, or an emulsion, i.e. ifthe drug is not substantially dissolved by diffusion or by beingsqueezed out by contraction of the fibrin gel formed. Such a fibrin geldepot may exert its activity for one week to two weeks or even longer.If, however, the drug is present in the form of a crystalloidalsubstance, it is usually dissolved and/or squeezed out of the fibrin gelwithin one day to two days while the fibrin gel remains in the body fora longer period of time. The same is true for other systems. Difiusionof a crystalloidal drug is also dependent upon the compactness of thegel.

The prolonged effect of a depot, according to the present invention, isfurthermore dependent upon other factors, such as injected volume, shapeof the gel within the tissue, i.e. its surface, place of injection inrelation to the flow of the body fluids, etc. Repeated application mightcause more rapid dissolution of a gel. When using the systemgelatin-formaldehyde or other hardening agents, the prolonged effect ofthe gel increases with increasing concentration of gelatin and/orformaldehyde. When producing a depot by intramuscular injection, it isadvisable to employ higher concentrations of the hardening agent becausein this instance, the gel is more rapidly dissolved. Formaldehyde gelsremain effective for days but quinone gels for weeks.

As stated above, when using a liquid and a solid component in separateampoules, the liquid, immediately before injection, is added to thesolid or, vice versa, the solid to the liquid. Two liquid components mayeither be mixed by adding the one to the other and mixing them outsidethe syringe or both may be withdrawn from the ampoules and may be mixedwithin the syringe.

The following examples serve to illustrate the present inventionwithout, however, limiting the same thereto.

Example 1 2 cc. of an aqueous solution containing 0.4 g. of gelatin, 0.1g. of potassium thioeyanate, 14 mg. of calcium chloride, and, suspendedtherein, mg. of estradiol benzoate, are filled into ampoule A. Anotherampoule B contains 4 mg. of formaldehyde in 0.5 cc. of Water. Bothampoules are heated to about body temperature. The contents of theformaldehyde ampoule B are added to the contents of the other ampoule Aand the combined solutions are quickly but thoroughly mixed. The mixtureis withdrawn by means of a syringe and is immediately injected. At theplace of injection, a depot of the estro enic hormone is formed due tohardening of the gelatine which takes place Within a few minutes.

13 Example 2 Ampoule A: 0.3 g. of meperidine hydrochloride dissolved in2 cc. of a 17% gelatin solution containing 0.2 g. of potassiumthiocyanate.

Ampoule B: 0.4 mg. of chromium in the form of 4 mg. of chrome alum and50 mg. of sodium chloride are dissolved in 0.5 cc. of water.

A depot of merperidine is produced by proceeding in the same manner asdescribed in the preceding example.

Example 3 Ampoule A: 0.3 g. of meperidine hydrochloride are dissolved in5 cc. of water. The pH-value of said solution is adjusted to a pH of 7.0by the addition of sodium hydroxide solution. The resulting solution isfilled up to 5 cc.

Ampoule B contains 0.3 g. of fibrinogen in its dry powdered form.

Ampoule C: 10 units of thrombin in dry powdered form.

The contents of ampoule A are added to the fibrinogen ampoule and shakenuntil all the fibrinogen is dissolved. Thereafter the solution is heatedto body temperature and is added to ampoule C containing the thrombinand is shaken until a clear solution is obtained. The mixture isimmediately injected, producing a depot of meperidine of prolongedactivity.

Example 4 Ampoule A: 0.5 g. of meperidine base are suspended in 5 cc. ofa 5% gelatin solution. The pH-value of said suspension is adjusted toabout 8.0 by the addition of sodium hydroxide solution.

Ampoule B contains 0.2 g. of fibrinogen and 2.5 units of thrombin, bothsubstances in dry powdered form.

Shortly before injection the contents of a-rnpoule A are added to thedry powder of ampoule B. The mixture is shaken until the fibrinogen andthrombin are completely dissolved, and is then injected. A depot ofmeperidine of prolonged activity is produced thereby.

Example Ampoule A contains 5 cc. of a phosphate buffer solution of a pHof 7.8.

Ampoule B contains 0.3 g. of fibrinogen in dry powdered state.

Ampoule C contains 5 units of thrombin and 1.0 g. of meperidine base.

First the contents of ampoule A are added to ampoule C until thethrombin is dissolved and themeperidine base is finely suspendedthroughout the solution. Said suspension, immediately before injection,is added to the contents of ampoule B and is thoroughly mixed therewithwhereafter the suspension is injected. A rneperidine depot of prolongedactivity is produced thereby.

Example 6 Ampoule A: 0.08 g. of morphine base are suspended in 2 cc. ofa 20% gelatin solution containing 15 mg. of calcium chloride.

Ampoule B: 0.5 mg. of chromium corresponding to 5 mg. of chrome alum.

Shortly before injection, the chrome alum in ampoule B is dissolved in0.5 cc. of water. The solution is added to the suspension of ampoule Aand is thoroughly mixed therewith. The ampoules were previously heatedto about 36 C. As soon as complete mixture is achieved, the suspensionis injected forming a morphine depot of prolonged activity.

Example 7 Ampoule A: 0.1 g. of morphine base are suspended in 3 cc. of a16% gelatin solution containing 90 mg. of potassium thiocyanate and 15mg. of calcium chloride.

Ampoule B: 0.6 mg. of chromium corresponding to 14 6 mg. of chromicalum, 4 mg. of formaldehyde, and 25 mg. of sodium chloride, dissolved in0.5 cc. of water.

The two solutions are withdrawn into a syringe wherein they arethoroughly mixed. On injection, the mixture forms a morphine depot ofprolonged activity.

Example 8 Ampoule A: 0.2 g. of morphine base are suspended in 6 cc. of a5% gelatin solution being adjusted, by the addition of sodium hydroxidesolution, to a pH of about 7.8.

Ampoule B contains 0.3 g. of fibrinogen and 3 units of thrombin in drypowdered form.

The contents of ampoule A are added, immediately before injection, toampoule B and are thoroughly mixed therewith. Thereafter the mixture isinjected and forms a morphine depot of considerably prolonged activity.Preparations containing morphine or other narcotics are of considerableimportance inasmuch as their depot effect prevents the patient reachingthe euphoric stage. As a result thereof the danger of addiction isconsiderably reduced.

Example 9 Amponle A: 0.5 in 2 cc. of a 15% gelatin solution containing0.1 potassium thiocyanate.

Ampoule B: 0.5 mg. of chromium corresponding to 5 mg. of chrome alum,dissolved in 0.5 cc. of water.

The two solutions are withdrawn from their ampoules into a syringe andare thoroughly mixed therein. After injection, a depot of codeinephosphate of considerably prolonged activity is obtained.

g. of codeine phosphate are dissolved g. of

Example 10 Example 11 Ampoule A: 100 mg. of dibucaine hydrochloride aredissolved in 2 cc. of a phosphate buffer solution having a pH of about7.0. 10 mg. of glycerol are added to said solution.

Ampoule B: 100 mg. of fibrinogen, about 2 units of thrombin, :and 25 mg.of sodium benzoate, all in dry powdered state. The contents of ampoule Aare transferred to arnpoule B' and are thoroughly rrnixed therewithuntil completely dissolved. Immediately thereafter, the mixture isinjected and forms a depot of the local anesthetic.

Example 12 Ampoule A: 2.2 cc. of a 1% solution of sodium benzoateadjusted to a pH of about 7.0.

Ampoule B: mg. of fibrinogen in dry powdered form.

Ampoule C: 1 unit of thrombin and 50 mg. of dibucaine hydrochloride,both substances in dry powdered form.

The contents of ampoule A are first added to ampoule B and shaken untilthe fibrinogen is dissolved. The solution is then added to ampoule C andagain shaken to dissolve the thrombin and local anesthetic agent. Themixture is immediately injected forming a depot of said local anestheticagent.

Example 13 Ampoule A: A 2% solution of dibucaine base in 2 cc. of oliveoil is emulsified in 5 cc. of a 5% gelatin solution.

Ampoule B: 7 units of thromb-in in dry powdered form.

Ampoule C: 0.2 g. of fibrinogen in dry powdered form.

Shortly before injection, ampoule A is mixed with em 15 poule B and themixture is transferred to ampoule C. Thereafter, the contents of saidampoules are thoroughly mixed and injected.

Example 14 Ampoule A: 10 cc. of a 10% gelatin solution containing 1.0 g.of procaine hydrochloride and being adjusted, by means of acetate buttersolution, to a pH of about 7.0.

Ampoule B: 200 mg. of fibrinogen in dry powdered form.

Ampoule C: 2.5 units of thrombin in dry powdered form.

The contents of ampoule A are added to ampoule B and, after fibrinogenis dissolved, to ampoule C. As soon as thrombin is dissolved, themixture is injected to form a depot of procaine. The gelatin addition tothe fibrinogen-thrornbin solution has the advantage that the gelconcentration is increased and the diffusion of procaine is furthermoreretarded.

Example 16 Ampoule A: 3 cc. of a 15% gelatin solution containing 150 mg.of procaine hydrochloride and 0.1 g. of potassium thiocyanate.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.7 mg. of chromiumin the form of 7 mg. of chrome alum and 0.1 g. of sodium chloride.

The solutions are mixed in a syringe and are injected immediately aftermixing, forming a deposit of procaine in the body. This preparation, onaccount of its chromium content, should not be used for nerve blockanesthcsia.

In a similar manner as described in the preceding examples, other localanesthetics may be used, such as butethamine hydrochloride or formate,butacaine sulfate, diperodon hydrochloride, lidocaine hydrochloride,naepaine hydrochloride, phenacaine hydrochloride, piperocainehydrochloride, and the bases derived therefrom, and others more. In thecase of anesthetic agents of the type of primary amines, it is advisableto use such solidifying or hardening agents which do not react with theprimary amino groups while with tertiary and secondary amines suchreactions are not to be feared.

Example 17 Ampoule A: 2 cc. of a 20% solution of camphor in peanut oilare emulsified in cc. of a 15% gelatin solution containing 35 mg. ofcalcium chloride.

Ampoule B: 1 cc. of an aqueous solution containing 0.1% of chromium inthe form of chrome alum.

Solution B is added, while stirring thoroughly, to emulsion A, therebyyielding a mixture which, on injection, produces a camphor depot in thebody.

Example 18 Ampoule A: 4 cc. of camphor liniment (US. Pharmacopoeia XIV)are emulsified in 4 cc. of a 12% gelatin solution containing 0.8 g. ofmagnesium chloride.

Ampoule B: 0.6 cc. of an aqueous solution of 1.0 mg. of chromium in theform of chrome alum.

The contents of ampoules A and B are mixed in a syringe and are theninjected, thereby forming a depot of camphor.

Example 19 Ampoule A: 1 g. of camphor is finely suspended in 5 cc. of agelatin solution.

Ampoule B: 0.2 g. of fibrinogen in dry powdered form.

Ampoule C: 5 units of thrombine in dry powdered form.

The camphor suspension of ampoule A is added to ampoule B. Afterfibrinogen is completely dissolved, the mixture is added to ampoule Cand is injected as soon as thrombine is dissolved, thereby forming acamphor depot.

Example 20 Ampoule A: 0.4 g. of methyl isopropyl cyclohexenone(hexetone) are emulsified in 3 cc. of a 16% gelatin solution containingmg. of potassium thiocyanate.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.6 mg. of chromiumin the form of chrome alum.

Both ampoule contents are mixed in a syringe and are injectedimmediately after mixing, forming a depot of said analeptic agent.

Example 21 Ampoule A: 2 cc. of a 16% gelatin solution containing 0.1 g.of potassium thiocyanate and 0.2 g. of pentylene tetrazole.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum.

Both solutions are mixed in a syringe and are then administered to forma pentylene tetrazole depot.

Example 22 Ampoule A: 3 cc. of a 16% gelatin solution containing 0.3 g.of pentylene tetrazole, 0.15 g. of a mixture of catieine and sodiumsalicylicum (1:1), and 90 mg. of potassium thiocyanate.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.7 mg. of chromiumin the form of chrome alum.

Mixing and administration of this preparation is carried out asdescribed in the preceding examples.

Example 23 Ampoule A: 10 cc. of a 20% gelatin solution containing 2.5 g.of nikethamide, 50 mg. of calcium chloride, and 0.3 g. of potassiumthiocyanate.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum. 5 ampoules B are provided for 1 ampoule A.

2 cc. of ampoule A are mixed in the syringe with 0.5 cc. of ampoule Band the mixture is then injected to form a nikethamide depot in thebody.

Example 24 Ampoule A: 4 mg. of strychnine nitrate are dissolved in 2 cc.of a 15% gelatin solution containing 0.2 g. of magnesium chloride.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum and 50 mg. of sodium chloride.

Both ampoule contents are mixed and administered as described in thepreceding examples to produce a strychnine depot.

Example 25 Ampoule A: 2 cc. of a 20% gelatin solution containingdigitalis glycosides corresponding to 0.2 g. of digitalis, and 0.2 g. ofmagnesium chloride.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum and 25 mg. of sodium chloride.

17 The two solutions are mixed and injected subcutaneously to form adepot of digitalis glycosides.

Example 27 Ampoule A: 0.1 mg. of ouabain, mg. of tetracainehydrochloride, and 0.1 g. of potassium thiocyanate are dissolved in 2cc. of a 15% gelatin solution.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum, and 25 mg. of sodium chloride.

Both solutions are mixed and administered as described in the precedingexamples to produce an ouabain depot.

Example 28 Ampoule A: 0.2 mg. of ouabain, mg. of dibucainehydrochloride, and 0.2 g. of magnesium sulfate are dissolved in 2 cc. ofan 18% gelatin solution.

Ampoule B: 0.5 cc. of an aqueous solution containing 0.3 mg. of chromiumin the form of chrome alum, 4 mg. of formaldehyde, and 50 mg. of sodiumchloride.

Both solutions are mixed in the syringe and immediately injectedsubcutaneously to produce a depot of ouabain.

Example 29 Ampoule A: 0.1 g. of heparin sodium and 60 mg. of potassiumthiocyanate are dissolved in 2 'cc. of a gelatin solution.

Ampoule B: 0.3 cc. of an aqueous solution containing 0.5 mg. of chromiumin the form of chrome alum.

Both solutions are mixed in the syringe and are then injectedsubcutaneously to produce a depot of heparin which slowly and graduallyreleases the heparin over a considerably more prolonged period of timethan heretofore possible.

Example 30 Ampoule A: 0.1 g. of bis-hydroxy coumarin and 30 mg. ofpotassium thiocyanate are dissolved in 2 cc. of a gelatin solution.

Ampoule B: 0.3 cc. of an aqueous solution containing 0.3 mg. of chromiumin the form of chrome alum and 15 mg. of sodium chloride.

Both solutions are mixed in a syringe and are then injectedintramuscularly to produce a depot of bis-hydroxy coumarin.

Other anticoagulants may be used likewise.

Example 31 Ampoule A: 300 mg. of methyl testosterone are suspended in 5cc. of a 5% gelatin solution containing 10 mg. of glycerol.

Ampoule B: 0.2 g. of fibrinogen in dry powdered form.

Ampoule C: 7.5 units of thrombin in dry powdered form.

The contents of ampoule A are added to ampoule B and, after fibrinogenis dissolved, to ampoule C, whereupon the mixture is injectedintramuscularly to form a depot of methyl testosterone.

Example 32 Ampoule A: 300 mg. of testosterone are finely suspended in 3cc. of a 15 gelatin solution containing 20 mg. of calcium chloride.

Ampoule B: 10 mg. of benzoquinone in dry powdered form.

The contents of ampoule A are added to arnpoule B and, after thoroughmixing, the mixture is injected to form a depot of testosterone.

Example 33 Ampoule A: 50 mg. of testosterone propionate dissolved in 1cc. of olive oil, are emulsified in 4 cc. of an 18% gelatin solutioncontaining 160 mg. of potassium thiocyanate.

Ampoule B: Contains 20 mg. of toluquinone.

18 First toluquinone is dissolved by adding 1.0 cc. of distilled andsterile water to ampoule B. To said solution the content of 'ampoule Ais given and rapidly and thoroughly mixed therewith. On injecting themixture, a depot of testosterone propionate is obtained.

Example 34 Example 35 Ampoule A: mg. of ethinyl estradiol are suspendedin 5 cc. of a 16% gelatin solution.

Ampoule B: Contains 24 mg. of benzoquinone.

The contents of arnpoule A are added to ampoule B and, afterbenzoquino-ne is dissolved, the mixture is injected to form a depot ofethinyl estradiol.

Example 36 Ampoule A: 100 mg. of diethyl stilbestrol dipropionate aresuspended in 6 cc. of a 10% gelatin solution containing 18 mg. ofglycerol.

Ampoule B: 0.2 g. of fibrinogen and 12 units of thrombine, bothsubstances in dry powdered form.

The suspension in ampoule A is added'to ampoule B and, after solutionhas taken place, the mixture is injected to form a diethyl stilbestroldipropionate depot.

Example 37 Ampoule A: 50 mg. of estradiol dipropionate are dissolved in1 cc. of sesame oil. Said solution is emulsified in 5 cc. of a 5%gelatin solution containing 25 mg. of glycerol.

Ampoule B: 0.3 g. of fibrinogen and 9, units of thrombin, bothsubstances in dry powdered form.

The contents of ampoule A are added to arnpoule B and the emulsionobtained on thorough mixing is injected to form a. depot of estradioldipropionate.

Example 38 Ampoule A: 50 .mg. of progesterone are dissolved in 1 cc. ofolive oil. Said solution is emulsified in 4 cc. of a 1% gelatinsolution.

Ampoule B: 0.2 g. of fibrinogen and 3 units of thrombin, both substancesin dry powdered form.

The contents of ampoule A are added to ampoule B and are mixedthoroughly therewith whereafter the mixture containing thedissolvedfibrinogen and thrornbin is injected'to produce a depot of progesterone.

Example 39 Ampoule A: mg. of progesterone are finely suspended in 6 cc.of a 10% gelatin solution containing 20 mg. of glycerol.

Ampoule B; 0.2 g. of fibrinogen in dry powdered form.

Ampoule C: 10 units of thrombin in dry powdered form.

The contents of ampoule A are added to and intimately mixed with thecontents of arnpoul-e B and the mixture thereof is added to andthoroughly mixed with the contents of ampoule C. The mixture is theninjected to form a depot of progesterone.

Example 40 Ampoule A: 250 mg. of progesterone are finely suspended in 3cc. of a 20% gelatin solution.

Ampoule B: 20 mg. of benzoquinone in dry powdered form.

19 The contents of ampoule A are added to ampoule B and are thoroughlymixed therewith. The mixture obtained is injected to form a depot ofprogesterone.

Example 41 Ampoule A: 50 mg. of desoxycorticosterone acetate are finelysuspended in cc. of a 20% gelatin solution.

Ampoule B: 20 mg. of benzoquinone in dry powdered form.

The contents of ampoule A are added to ampoule B and are intimatelymixed therewith until the benzoquinone is dissolved. Immediatelythereafter, the mixture is injected to form a depot ofdesoxycorticosterone acetate.

Example 42 Example 43 Ampoule A: 6 cc. of a phosphate bufier solutionhaving a pH-value of about 7.0.

Ampoule B: 0.4 g. of fibrinogen in dry powdered form.

Ampoule C: 2000 units of chorionic gonadotropin and 10 units ofthrombin, both substances in dry powdered form.

The contents of ampoule A are added to ampoule B and the fibrinogen isdissolved therein. The mixture is added to ampoule C and, after solutionhas taken place, is injected to form a depot of chorionic gonadotropin.

Example 44 Ampoule A: 5 cc. of a phosphate buffer solution having apH-value of about 7.0.

Ampoule B: 0.3 g. of fibrinogen and 5000 units of chorionicgonadotropin, both substances in dry powdered form.

Ampoule C: 1 cc. of the same buffer solution containing 20 mg. ofglycerol.

Ampoule D: 10 units of thrombin in dry powdered form.

First the contents of ampoules A and B are combined and the solidsubstances are dissolved. The contents of ampoules C and D are combinedto dissolve thrombin. Thereafter the two solutions are Withdrawn into asyringe and are intimately mixed therein. The mixture is then injectedto form a depot of chorionic gonadotropin.

Example 45 Ampoule A: 5 cc. of a phosphate bufi'er solution having apH-value of about 7.0 and containing mg. of glycerol.

Ampoule B: 0.3 g. of fibrinogen, units of vasopressin tannate, and 12.5units of thrombin, all these substances in dry powdered form.

The contents of ampoule A are added to ampoule B and are thoroughlymixed therewith. Thereafter, the mixture is injected and produces adepot of vasopressin.

Example 46 Ampoule A: 3 cc. of a buffer solution having a pH- va-lue ofabout 7.0 and containing 12 mg. of glycerol.

Ampoule B: 0.1 g. of fibrinogen, 2 units of thrombin, and 50 mg. ofadrenocorticotropic hormone of the pituitary gland, all these substancesin dry powdered form.

The contents of ampoule A are added to ampoule B and the mixture is,after complete solution has taken place, immediately injected forming adepot of said adrenocorticotropic hormone.

Example 47 Ampoule A: 0.05 g. of fibrinogen, 2 units of thrombin, and200 units of zinc insulin crystals (with about 0.75% of zinc), all thesesubstances in dry powdered form.

Ampoule B: 3 cc. of a phosphate bufier solution having a pH-value ofabout 7.0.

The contents of ampoule B are added to ampoule A and the mixture, afterthoroughly mixing, is injected to form an insulin depot of considerablyprolonged activity.

Example 48 Ampoule A: 1 cc. of a suspension of 300,000 units of procainepenicillin G in peanut oil is emulsified in 2 cc. of a 5% gelatinsolution.

Ampoule B: 0.2 g. of fibrinogen in dry powdered form.

Ampoule C: 6 units of thrombin in dry powdered form.

The contents of ampoule A are added to ampoule B. As soon as fibrinogenis dissolved, the mixture is added to ampoule C and is again intimatelymixed until thrombin is dissolved. Immediately thereafter, the mixtureis injected producing a depot of penicillin.

Example 49 Ampoule A: 4 cc. of a 2% procaine solution.

Ampoule B: 0.1 g. of fibrinogen, 2 units of thrombin, and 2,000,000units of procaine penicillin G, all these substances in dry powderedform.

The contents of ampoule A are added to ampoule B and are intimatelymixed therewith. Injection of the mixture produces a penicillin depot ofconsiderably prolonged activity.

Example 50 Ampoule A: 10 cc. of a 20% gelatin solution containing about0.5 g. of potassium thiocyanate.

Ampoule B: 3 g. of streptomycin sulfate in dry powdered form.

Ampoule C: 2.5 cc. of an aqueous solution containing 2.5 mg. of chromiumin the form of chrome alum and 250 mg. of sodium chloride.

Ampoules A and B are mixed with each other and the mixture is added toampoule C. After intimately mixing, the mixture is injected, producing adepot of streptomycin of considerably prolonged activity.

Example 51 Ampoule A: 2 cc. of a 10% gelatin solution.

Ampoule B: 250 mg. of aureomycin hydrochloride in dry powdered form.

Ampoule C: 50 mg. of fibrinogen and 1 unit of thrombin, both substancesin dry powdered form.

The contents of ampoules A and C are mixed with each other. The mixtureis added to ampoule B and vigorously shaken. On injecting the resultingmixture, a depot of aureomycin is produced.

Example 52 Ampoule A: 3 cc. of a 3% gelatin solution, being adjusted toa pH of about 7.0

Ampoule B: 0.1 g. of fibrinogen, 1.5 units of thrombin, 300,000 units ofcrystalline procaine penicillin G, 100,000 units of buffered crystallinesodium penicillin G, and 1 g. of dihydrostreptomycin in the form of itssulfate, all said compounds present in the form of a fine dry powder.

The contents of ampoule A are added to ampoule B and are vigorouslyshaken therewith. The mixture obtained is then injected intramuscularlyand forms a depot of the antibiotics of considerably prolonged activity.

Example 53 Ampoule A: 2 cc. of a 5% gelatin solution.

Ampoule B: 500 mg. of crystalline terramycin hydrochloride in drypowdered form.

Ampoule C: 50 mg. of fibrinogen and 1 unit of thrombin, both in drypowdered form.

The contents of ampoulcs A and C are mixed with each other and, afterfibrinogen and thrombin are dissolved, the solution obtained is added toampoule B. The mixture is vigorously shaken and is then injectedintramuscularly to produce a depot of the antibiotic of considerablyprolonged activity.

Example 54 Ampoule A: cc. of a gelatin solution containing 0.2 g. ofpotassium thiocyanate and finely suspended therein 1 g. of iodoform.

Ampoule B: 58 mg. of dichro chloride, g. of silver thiocyanate, and mg.of barium sulfate.

Ampoule C: 1.5 cc. of a 5% solution of ammonium chloride.

The contents of ampoule C are added to ampoule B to dissolve the dichrochloride and to suspend the insoluble salts by repeated vigorousshaking. The mixture is allowed to stand for about 30 minutes. Saidsuspension is then added to ampoule A and is thoroughly mixed therewith.After tWo minutes the mixture is introduced into an abscess or a fistulawherein it solidifies to a. gel which slowly and continuously releasesthe disinfecting agent.

. Example Ampoule A: 5 cc. of a 15% gelatin solution containing 0.5 g.of magnesium chloride, and 0.5 g. of the sodium salt of p-aminosalicylic acid.

Ampoule B: 1 cc. of an aqueous solution containing 1 mg. of chromium inthe form of chrome alum and 50 mg. of sodium chloride.

The contents of both ampoules are intimately mixed in a syringe and thenintroduce into abscesses caused by tuberculosis. The solidified gelslowly and gradually releases the pamino salicylic acid.

Example 56 Ampoule A: 10- cc. of a 12% gelatin solution containing 2 g.of ethylstibamine, 40 mg. of barium sulfate and 5 mg. of dibucainehydrochloride.

Ampoule B: 70 mg. of benzoquinone in dry powdered form.

The contents of ampoule A are added to ampoule and are intimately mixedtherewith until benzoquinone rs completely dissolved. The mixture isthen injected ntramuscularly to form a depot of ethylstibamine having aprolonged effect.

Of course, many changes and variations may be made in the above givenexamples. Other therapeutically effective. compounds may be usedprovided it is desired to produce a prolonged effect of the same. Apreparation according to this invention may have the further advantagethat the drug is released at about the same rate over almost the entireperiod of activity while with depots as they were employed heretofore,usually a very strong shock-like effect was initially observed whilesubsequently the activity of said depot diminished considerably andfrequently did not suffice to produce satisfactory therapeutic effects.Other hardenable, coagu-lable, settable, solidifiable, or gellingcompounds and other agents causing such hardening, coagulating, setting,solidifying, or gelling than those mentioned in the examples may also beused. Likewise, other agents to regulate viscosity, to retard oraccelerate hardening, coagulating, setting, solidifying, or gelling, tostabilize the compositions according to the present invention and theircomponents, to ad ust their pH-value, to protect the therapeuticallyeffective compounds, than those mentioned in the examples may be added.Many changes in the temperature, the reaction conditions,concentrations, and proportions of the com ponents may be made by thoseskilled in the art in accordance with the principles set forth hereinand in the claims annexed hereto.

The term protein-like compound, the aqueous solution of which issolidified by the action of a chemical solidi? fying agent, as used inthe specification and the claims annexed hereto, includes not onlyproteins such as gelatin, but also other hardenable, coagulab-le,settable, solidifiable, gelling compounds, such as fibrinogen,hyaluronic acid, nucleic acids, certain polysaccharides, vegetable gums,synthetic resinous compounds, and others more, provided their hardening,coagulating, setting, solidifying, or gelling time can be retarded oraccelerated to such an extent that their aqueous solutions can safely beinjected in the fluid state but form, shortly after injection, ahardened, coagulated, set, solidified, or gelled depot in the body whichis slowly and gradually absorbed by the body and the body fluids andwhich, thus, slowly and gradually releases the therapeutically effectivecompound uniformly distributed therethrough and, thereby causing saidtherapeutically effective compound to exert a prolonged therapeuticeffect.

The terms solidified, solidifying, solidifiable, and the like as used inthe specification and the claims an-. nexed hereto, refer not only tothe conversion of a substance or substance mixture into the solid state,but indicate also any other conversion from the fluid into the solidstate, such as hardening, coagulating, setting, gelling, and othersmore. solidified depots according to this invention are relativelyinsoluble in water but they are slowly and gradually dissolved,absorbed, decomposed, or in any other manner eliminated by the bodyfluids on account of their enzymatic or otherwise dissolving power.

The term aqueous medium, as used in the specification and claims annexedhereto, includes not only water, but also other water-containing media,such as aqueous salt solutions, aqueous buffer solutions, emulsions ofthe water-in-oil and the oi-l-in-water type, or any other aqueous mediumcapable of dissolving the solidifiable compound and the solidifyingagent and being injectable and substantially non-toxic to the body.

The term therapeutically effective compound as used in the specificationand in the claims annexed hereto, indicates not only drugs used intherapy for curing and healing but also chemical compounds which areused as auxiliary means in combatting diseases, such as anesthetics,diagnostic aids, X-ray contrast agents, disinfectants, antiseptics,compounds used for nutritional purposes, prophylatics and others more.Likewise the term therapeutic effect indicates the effects of suchso-called therapeutically effective agents.

The present invention can also be used with great advantage in theadministration of orally effective agents which ordinarily produce apronounced initial effect that may be harmful to certain sensitivepatients. Injection of the depot preparations according .to the presentinvention is accompanied by a gradual and uniform release of thetherapeutic agent and thus avoids any harmful initial or shock-likeeffect.

We claim:

1. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninject-able aqueous medium, fibrinogen dissolved therein, a watersoluble compound forming fibrin with fibrinogen, and the drug to beadministered uniformly distributed throughout the composition, saidcomposition being liquid at body temperature for a time sufiicient forinjection and, upon injection into the human and animal tissue,depositing said drug depot at the site of injection.

2. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, fibrinogen dissolved therein, thrombin, andthe drug to be administered uniformly distributed throughout thecomposition, said composition being liquid at body tem- 23 perature fora time sufiicient for injection and, upon injection into .the human andanimal tissue, depositing said drug depot at the site of injection.

3. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, gelatin dissolved therein in an amount of atleast of said composition, a water soluble chromium salt causingcoagulation of said gelatin to a gel, and the drug to be administereduniformly distributed throughout the composition, said composition beingliquid at body temperature for a time sufiicient for injection and, uponinjection into the human and animal tissue, depositing said drug depotat the site of injection.

4. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, gelatin dissolved therein in an amount of atleast 10% of said composition, a water soluble chromium salt causingcoagulation of said gelatin to a gel, said gelatin and said chromiumsalt being present in said composition in the proportion between about 1part of chromium ion to about 130 parts of gelatin and about 1 part ofchromium ion to about 1350 parts of gelatin, and the drug to beadministered uniformly distributed .throughout the composition, saidcomposition being liquid at body temperature for a time suflicient forinjection and, upon injection into the human and animal tissue,depositing said drug depot at the site of injection.

5. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, fibrinogen dissolved therein in an amountcorresponding to at least about 1.7% of the composition, thrombin, saidfibrinogen and said thrombin being present in said composition in theproportion between about 1 unit of thrombin to about 75 mg. offibrinogen and about 1 unit of thrombin to about 100 mg. of fibrinogen,and the drug to be administered uniformly distributed throughout thecomposition, said composition being liquid at body temperature for atime sufficient for injection and, upon in jection into the human andanimal tissue, depositing said drug depot at the site of injection.

6. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, gelatin dissolved therein in an amount of atleast 10% of said composition, a mononuclear aromatic quinone, and thedrug to be adminstered uniformly distributed throughout the composition,said composition being liquid at body temperature for a time sufficientfor injection and, upon injection into the human and animal tissue,depositing said drug depot at the site of injection.

7. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, gelatin dissolved therein in an amount of atleast 10% of said composition, benzoquinone, and the drug to beadministered uniformly distributed throughout the composition, saidcomposition being liquid at body temperature for a time suflicient forinjection and, upon injection into the human and animal tissue,depositing said drug depot at the site of injection.

8. An injectable pharmaceutical composition forming, on injection, adrug depot at the site of injection, said composition comprising aninjectable aqueous medium, gelatin dissolved therein in an amount of atleast 10% of said composition, a Water soluble metal salt causingcoagulation of said gelatin to a gel, and the drug to be administereduniformly distributed throughout the composition, said composition beingliquid at body temperature for a time sufiicient for injection and, uponinjection into the human and animal tissue, depositing said drug depotat the site of injection.

9. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousfibrinogen solution of at least 1.7% of fibrinogen with a water solublecompound forming fibrin with fibrinogen and incorporating into anduniformly distributing throughout said mixture the drug to beadministered by injection, the amount of said fibrinforming compoundbeing insufiicient to form fibrin during mixing and injection butsufiicient to deposit the drug and the formed fibrin in the form of adrug depot at the site of injection.

10. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousfibrinogen solution of at least 1.7% of fibrinogen with thrombin andincorporating into and uniformly distributing throughout said mixturethe drug to be administered by injection, said fibrinogen and saidthrombin being present in said mixture in the proportion between about 1unit of thrombin to about mg. of fibrinogen and about 1 unit of thrombinto about mg. of fibrinogen.

11. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousgelatin solution of at least 10% gelatin with a water soluble chromiumsalt causing coagulation of said gelatin to a gel and incorporating intoand uniformly distributing throughout said mixture the drug to beadministered by injection, the amount of said chromium salt beinginsufiicient to cause coagulation of the preparation during mixing andinjection but sulficient to deposit the drug and the coagulated gelatinin the form of a drug depot at the site of injection.

12. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousgelatin solution of at least 10% gelatin with a water soluble chromiumsalt causing coagulation of said gelatin to a gel and incorporating intoand uniformly distributing throughout said mixture the drug to beadministered by injection, said gelatin and said chromium salt beingpresent in said mixture in the proportion between about 1 part ofchromium ion to about parts of gelatin and about 1 part of chromium ionto about 1350 parts of gelatin.

13. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousgelatin solution of at least 10% gelatin with a water soluble metal saltcausing coagulation of said gelatin to a gel and incorporating into anduniformly distributing throughout said mixture the drug to beadministered by injection, the amount of said metal salt beinginsulficient to cause coagulation of the preparation during mixing andinjection but sufiicient to deposit the drug and the coagulated gelatinin the form of a drug depot at the site of injection.

14. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousgelatin solution of at least 10% gelatin with a water solublemononuclear aromatic quinone and incorporating into and uniformlydistributing throughout said mixture the drug to be administered byinjection, the amount of said quinone being insuflicient to causecoagulation of the preparation during mixing and injection butsufiicient to deposit the drug and the coagulated gelatin in the form ofa drug depot at the site of injection.

15. In a method of producing an injectable pharmaceutical preparation,the steps comprising mixing, shortly before injection, an aqueousgelatin solution of at least 10% gelatin with a water solublebenzoquinone and incorporating into and uniformly distributingthroughout said mixture the drug to be administered by injection, theamount of said benzoquinone being insufficient to cause coagulation ofthe preparation during mixing and injection but sufiicient to depositthe drug and the coagulated gelatin in the form gf a drug depot at thesite OTHER RE ERENCES l' Progress in Leather Science, 1929-1945, BritishLeather References Cited in the file of this patentlfgilglylfggtglresrgglResearch Association, London, England, UNITEDSTATES PATENTS 5 OFlaherty et a1.: Chemistry and Technology of 2 530 430Pi ki N 21, 1950 Leather, v01. 2, Reinhold Publishing Corp., New York,

2,533,004 Ferry et a1. Dec. 5, 1950 1958, p g

1. AN INJECTABLE PHARMACEUTICAL COMPOSITION FORMING, OR INJECTION, ADRUG DEPOT AT THE SITE OF INJECTION, SAID COMPOSITION COMPRISING ANINJECTABLE AQUEOUS MEDIUM, FIBRINOGEN DISSOLVED THEREIN, A WATER SOLUBLECOMPOUND FORMING FIBRIN WITH FIBRINOGEN, AND THE DRUG TO BE ADMINISTEREDUNIFORMLY DISTRIBUTED THROUGHOUT THE COMPOSITION, SAID COMPOSITION BEINGLIQUID AT BODY TEMPERATURE FOR A TIME SUFFICIENT FOR INJECTION AND, UPONINJECTION INTO THE HUMAN AND ANIMAL TISSUE, DEPOSITING SAID DRUG DEPOTAT THE SITE OF INJECTION.